Studies of the oxidative demethylation at C4 which occurs during the latter stages of cholesterol biosynthesis are being continued. Tritium-labeled synthetic steroidal substrates, designed to test geometric or electronic aspects of the demethylation process, are mixed with rat liver homogenate enzyme preparations, and the amount and nature of the radioactive products formed are determined. Efforts are also in progress to simulate the enzymic process by developing a laboratory method for selective oxidation of the C4 methyl groups in 3-oxygenated steroids.